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Learning objectives After studying this chapter you should confidently be able to:
Describe why sections need to be coloured with dyes. Staining is needed to give contrast between different components of the tissues and allow examination by light microscopy. Describe how dyes bind to tissues. Dyes bind by forming bonds with tissue components. Ionic and hydrogen bonding and van der Waals forces are probably all involved. Ionic staining is the most important and distinguishes between basophilic and acidophilic tissue components. Hydrogen bonding and van der Waals forces are less important but probably play a role in selectivity. Describe the use of mordants in staining. Mordants are metal salts that help bind some dyes to tissues. Haematoxylin is the most important mordanted dye. Define metachromasia and give examples of its use. Metachromasia produces a different colour in a tissue component to the colour of the dye solution. Toluidine blue is blue in solution but stains mast cell granules red. Describe the main properties of haematoxylin and staining using haematoxylin solutions. Haematoxylin is a natural dye that requires oxidation to haematein before use as a stain. Haematoxylin is a mordanted dye that can stain many different elements in tissue depending on the mordant used. Using different mordants it can be used to stain nuclei, connective tissue fibres, nerve cells, muscle striations and mitochondria. It is usually used regressively. Describe the use of silver as an impregnating metal. Silver solutions are easily reduced producing a dense black deposit and this reduction is autocatalytic. In argentaffin reactions, no extra reducing agent is needed, but argyrophil reactions require the addition of a reducing agent. Describe the reasons for mounting tissues and outline the types of mounting media. Mounting media and coverslips not only protect the specimen but also make it translucent, making examination easier. Mounting media may be resinous (organic-based) or water-based solvents.
68 Chapter 6
If sections of human tissue are examined under the microscope immediately after sectioning, they appear very dull and uninteresting. The tissue lacks contrast because all of the fixed materials have a similar refractive index and a similar colour so that a dull grey colour is all that can be seen. To bring out the structure of the tissues, it is essential to stain the cells to see the different parts in contrasting colours. Staining is not simply random colouring of the sections but depends on using differences in the chemistry of the tissue to show the various components in different colours. This is most commonly done using dyes that can bind to the tissue in a selective way. Thus, the colours that are seen reflect the nature of the tissue and are not just a pretty picture. By using two or more dyes, it is possible to bring out the different materials in several contrasting colours. The commonest stain in use is the haematoxylin and eosin (H&E) stain, which colours the nuclei a dark blue or purple and stains the cytoplasm and connective tissue in shades of pink (see Colour plate 5).
The binding of dyes to tissues is no different to any other chemical bonding and the mechanisms rely on the same binding forces that occur in all other organic compounds. The dye must form some type of bond or link to the tissue or they will simply be rinsed out of the tissue when the section is washed in another reagent. The usual forms of bonding can be involved. Each type has its own characteristics and bond strengths. Bond type Ionic bonds Hydrogen bonds Van der Waals forces Covalent bonds Hydrophobic interactions Ionic bonding Ionic bonding involves electrostatic attraction between oppositely charged ions. One ion is a fixed ion in the tissue section and the other is the dye ion. Anionic (negatively charged) dyes will bind to cations (positively charged) in the tissue, and cationic dyes will bind to tissue anions. Ionic bonding is the single most important form of bonding in most histological staining. Almost all simple staining can be understood and controlled by understanding the ionic charges involved. Negatively charged eosin ions will stain positively charged tissue ions Eosin is an example of an anionic dye and is attracted to protein groups that are positively charged (cations) such as amino groups. First, the amino Strength (kcal mole–1) 40–110 2–7 1-2 35–212 4–8
6.1 Staining mechanisms
Figure 6.1 The ionization of tissue amines and subsequent binding of eosin
groups in the protein become ionized by binding to a hydrogen ion and this charged group then attracts the eosinic ion (see Fig. 6.1). Eosin is usually sold as a salt such as sodium eosinate, which is readily soluble in water. Anionic dyes are also called acid dyes in histology because they are derived from coloured acids (in this case, eosinic acid) and not because of the pH of the solution. Anything that will stain with an acid dye is called acidophilic. In the case of materials staining with eosin, they could also be termed eosinophilic. Materials that are acidophilic include collagen, red blood cells and the cytoplasm of many cells.
Box 6.1 Controlling pH allows dyes to be removed Sensitivity to pH is exploited in the removal of dyes. This is used in differentiation and is also important in getting dye splashes off skin and clothing. Basic dyes can be removed by using acids (usually acid alcohol), whilst acidic dyes are most easily removed with a solution of ammonia in alcohol. These should always be very dilute solutions and used with extreme care. The solutions are extremely painful if used on cuts or sensitive areas such as around the eyes. It is much better to avoid getting the dye on your skin in the first place.
Positively charged methylene blue ions will stain negatively charged tissue ions Methylene blue is an example of a cationic dye and will bind to tissue anions such as carboxylic acid, sulphuric acid and phosphoric acid groups. These groups need to be ionized to bind the dyes (see Fig. 6.2).
OSO3 Methylene blue
Figure 6.2 The ionization of sulphate groups in mucins and their subsequent binding of methylene blue
the high concentration of hydrogen ions favours the ionization of amino groups and results in strong staining of proteins by eosin. which uses potassium hydroxide to raise the pH. This is most apparent in the staining of mucins where pH is used to control the binding of alcian blue to the weak carboxylic acid-containing mucins and the strong sulphated mucins (see Chapter 7). Alkaline conditions favour staining with cationic dyes Alkaline solutions will have the opposite effect. it is possible to get highly selective staining of individual components. At these acid pH levels. but total inhibition of ionization of the salt forms of dyes will only occur at extreme pH levels. To get maximal staining with methylene blue. In practice this does not occur because most staining is done at a neutral or slightly acid pH. At an acidic pH. Altering the pH can inhibit dyes from ionizing. Eosin staining is depressed at high pH since the amino groups are now unionized and no longer attract and bind the eosin.3). Since there are two different charges. as described above. Acid pH levels favour staining with anionic dyes Ionic bonding to dyes and therefore staining is pH sensitive since the ionization of tissue groups is affected by pH. are less easily inhibited and will still ionize at the pH levels generally used in staining. it is best to use Löffler’s formula. Ionic interactions are long-range forces and can attract dyes to tissues over relatively large distances. Methylene blue will then stain all of the proteins and the nucleic acids so the whole of the tissue will appear blue and there will no longer be any differential staining. Stronger acids.70 Chapter 6 Staining theory Cationic dyes are commonly called basic dyes and so substances staining with such dyes are called basophilic. Binding of dyes depends on tissue ionization Proteins normally contain both acidic and basic amino acids and so it might be expected that proteins would take up both dyes. Dyes will only bind to tissue groups when they are ionized. such as the phosphate groups found in nucleic acids and sulphate groups found in mucins. the same acidic pH will have the opposite effect on staining by methylene blue since weak acids. The ionization of dyes can be assumed to be complete at normal staining pH levels. By careful selection of the pH. they can repel as well as attract (see Fig. 6. However. picking out the nuclei but leaving the proteins unstained. such as the carboxylic acids found in proteins. The lack of hydrogen ions will allow the weakly acidic groups in proteins to ionize and methylene blue will stain both the cytoplasm and the nucleus. . will be inhibited from ionizing by the high concentrations of hydrogen ions. if the groups are unionized they will not attract the dye ions and will remain unstained. This means that in slightly acid solutions methylene blue will act as a differential stain. Substances that bind basic dyes include nucleic acids and acid mucins. the carboxyl groups of most amino acids are not ionized.
6. The salt may enhance staining slightly since both ions will be interacting. 6.3 Effect of opposite charges. In many ways this resembles the effect of acids on basic dyes where the hydrogen ion is competing for binding with the dye molecule. Although there is a negatively charged carboxyl group in the tissue that could bind the dye. The net charge on the surface of the protein is negative .1 Staining mechanisms 71 T I S S U E NH3 COO NH3 Methylene blue Figure 6. so the dye may be able to bind to sites to which it was previously not attracted due to the salt ions masking a repelling charge (see Fig. so the methylene blue ion is repelled Salt concentrations affect dye staining Ionic binding can be inhibited by high salt concentrations. the dye will not be attracted to it because of the two nearby positive charges. This paradoxical effect of both enhancing and inhibiting dyeing can be understood by the effects of the ions. although weak salt solutions can act to increase the staining. In strong salt solutions.4 Effect of ‘salting on’. Hydrogen bonding Hydrogen bonding differs from other uses of the word ‘bond’ since it is a force of attraction between a hydrogen atom in one molecule and a small T I S S U E NH3 COO NH3 Cl Methylene blue Cl Figure 6.3. Salt will affect both acid and basic dyes. there will be competition between the dye and the salt ions so staining will be inhibited. the binding of the dye will be stronger than the binding of the salt.4). Compare this with Fig. Low concentrations of salt will be less inhibitory since there will be more dye than salt and once a dye has bound. since the repelling charges on the amino groups are temporarily neutralized with chloride ions. 6. The net charge on the surface of the protein is positive. The methylene blue is now able to bind to the carboxyl group.
only a few atoms will be close enough to form van der Waals forces and the dye will be less strongly bound . If they are further apart. If the dye and tissue have similar shapes.72 Chapter 6 Staining theory atom of high electronegativity in another molecule. more atoms can be brought close enough together to form van der Waals forces and this can add up to a significant binding force. Hydrogen bonds are highly selective as they can occur only between certain groups (one must act as a donor and the other as a recipient). although they are individually very T I S S U E D Y E T I S S U E D Y E Van der Waals bonds Figure 6. Hydrogen bonding is not affected by pH or salt concentration but is affected by strong hydrogen-bonding agents including urea and water. Thus. then there is no effective bonding force. then many van der Waals bonds can be formed. If the surface shape of the tissue protein and the shape of the dye match. Van der Waals forces These are short-range forces and will only have an effect if the two atoms are between about 0. One or two instances of staining involve hydrogen bonding rather than ionic bonding. More controversially the staining of amyloid by Congo red has been considered to be dominated by hydrogen-bond staining. it is an intermolecular force.5 Van der Waals forces and dye binding. In the staining of elastin fibres hydrogen bonds are probably more important than ionic forces. They probably play a role in the selectivity of dyes. Van der Waals forces can occur between any two atoms and are not specific for any atom or group.2 nm apart.12 and 0. This inhibition of ionic bonds can be achieved by using a non-aqueous solvent (water inhibits hydrogen bonding and favours ionization). If the dye and tissue shapes are not complementary. not an intramolecular force as in the common use of the word bond. a high salt concentration (which competes with the dye for tissue ion-binding sites) and an extreme pH (which is chosen to inhibit the ionization of tissue groups). The hydrogen bond has a very limited range and will only form if the two interacting groups are brought sufficiently close together. but are usually secondary to ionic bonds unless special conditions are arranged to inhibit ionic interactions. Thus.
and in the attachment of dyes to antibodies in immunofluorescence. usually . Hydrophobic interactions are important in selectivity and play a major role in the staining of lipids. the forces are not chemical bonds in the conventional sense since they hold dyes in tissues by the exclusion of water from the regions of hydrophobic groups. The exclusion of water stabilizes the two groups involved by entropy/enthalpy changes. Hydrophobic interactions again are short range and are unaffected by hydrogen-bonding agents or salts. 6. With the use of silane-treated slides. The so-called reactive dyes use covalent bonds to bind but are not used much in histology. Covalent bonds These are very strong bonds and are not easily broken once formed. even though they may have the same number of ionizable groups. 6. Van der Waals forces are believed to have a role in selectivity but probably only play a minor role in actually binding of the dye to the tissue. they may add up to a significant binding force (see Box 6. The ability to form many van der Waals bonds is one explanation of the finding that larger dyes will bind more strongly than small dyes. Van der Waals bonds are unaffected by pH.g. periodic acid–Schiff. ions and hydrogen-bonding agents.6. Millions of van der Waals interactions are brought about and the section becomes firmly adherent. there is the addition of charge to the surface resulting in ionic bonding and the increased strength of adhesion may well reflect the difference in strength of the two bonds. weak. As the water evaporates from under the section. Hydrophobic interactions Although they are sometimes called hydrophobic bonds.2 Van der Waals forces are important in section adhesion Van der Waals forces are important in staining but in a quite different way to the binding of the dye.5). They are important in some histochemical techniques e. They do not seem to be important in most staining reactions. the lower surface of the section comes into contact with the flat smooth surface of the slide.2 Dye structure 73 Box 6. Most modern dyes are synthesized from simpler organic molecules.2) if the dye and protein have complementary molecular surfaces (see Fig. The adhesion of the section to the slide involves van der Waals interactions between the section and the glass.2 DYE STRUCTURE Dyes are coloured organic compounds that can selectively bind to tissues. Altering the pH may change a particular group from a hydrophilic to a hydrophobic form by altering its ionization and this will alter the staining with hydrophobic dyes.
The dye Tyrian purple was therefore a major discovery. W. is still not a dye. Perkin was a chemistry student trying to make the drug quinine from coal tar when he found some bright purple crystals. Benzene must be altered so that it will absorb visible light and so become a visible coloured compound that can be a useful as a dye. Legend has it that this dye was discovered by the demigod Hercules. Benzene. adding a second and third group intensifies the yellow colour and trinitrobenzene is a strong yellow colour. Benzene can be made to absorb visible light by adding a suitable chromophore. It is extracted from molluscs and by ancient standards was bright and did not fade quickly.H. Any group that makes an organic compound coloured is called a chromophore. In the example below (see Fig. the only dyes available were natural ones. Auxochromes Trinitrobenzene. benzene and alcohols are colourless to the human eye but will absorb light outside the visible spectrum. Chromophores Most simple organic compounds such as alkanes. The addition of an ionizable OH group turns trinitrobenzene into the dye trinitrophenol.74 Chapter 6 Staining theory Box 6. Treating the section with trinitrobenzene will temporarily colour it yellow in the same way that a plastic sponge appears coloured when it is soaked in a coloured liquid but the colour will wash out as soon as the tissue is rinsed in a solvent.6). The discovery of the first synthetic dye.H. for example. was entirely accidental. although coloured. which is a pale yellow colour. It became a symbol of power and wealth in the ancient world and the dictionary still lists purple as meaning wealth and power. Adding a single nitro group gives nitrobenzene. Picric acid is an acid dye (the OH group is phenolic and ionizes by losing a hydrogen ion) and is very useful . He borrowed some money and set up a factory to produce the dye and was rich enough to retire at 35. as it will not bind to tissues. benzene or one of its derivatives (see Box 6.3). but a simple example will show the general nature of dye structure. absorbs strongly in the UV region of the spectrum but appears water-white to the human eye. These tended to be somewhat drab and faded easily. To turn a coloured compound into a dye requires the addition of an ionizable group that will allow binding to the tissues. the chromophore used is the nitro group. Such binding groups are called auxochromes. The modification of these compounds into dyes is a huge industry and the chemistry of dye synthesis can be complex. Perkin in the 19th century. 6.3 Synthetic dye discovery Until the discovery of the first synthetic dye by W. which is more commonly called picric acid in histology. Mauve.
7 Quininoid structure. Figure 6.2 Dye structure NO2 2ON 75 NO2 Chromophore Benzene (colourless) Trinitrobenzene (yellow but not a dye) NO2 2ON NO2 Auxochrome OH Trinitrophenol (picric acid. This has two double bonds at either end of the ring and two double bonds on either side. It was originally discovered by oxidizing indigo. It is used in histochemical tests to dissolve and thus identify the pigments found in malarial cells and following formalin fixation. Picric acid is an unusual material in histological terms since it is a good fixative and forms the basis of the highly rated Bouin’s fixative.7). a yellow dye) Figure 6.6 Conversion of benzene into a dye by the addition of a chromophore and an auxochrome in histology (see Box 6.6. The most important chromophoric group in dye structure is not the nitro group but the quininoid arrangement of the benzene ring (see Fig.4). a natural dye from an Asiatic plant. 6. it is an essential part of the popular van Gieson counterstain. This arrangement strongly absorbs parts of the visible light spectrum. .4 Picric acid Picric acid is a powerful explosive and needs to be kept moist. It was known as Lyddite after the explosive works at Lydd where it was manufactured. an important chromophore Box 6. and it is also a useful dye because it has a low molecular weight.
it is usually easy to check with the supplier who will be able to put your mind at rest. a dye called light green is usually considered an acid dye in histology and used for staining connective tissue. The common name is easier to remember and to say. The full chemical name of this structure is 3. Although a dye may have more than one name.8 illustrates the complexity of many of these dyes. bromoacid TS. as well as allowing the dye to bind to the tissue. so a wide variety of different dyes have been synthesized to give a large range of colours. Eosin Y (yellowish eosin) has the following alternative names: acid red 87. bromoacid J. bromofluorescein and bronze bromo. which is the same dye by a different name. The dye structure shown in Fig. Modifiers do not greatly alter the staining characteristics of the molecule. Other groups or atoms are introduced solely to alter the colour and these are called modifier groups. 6.76 Chapter 6 Staining theory Changing substituent groups The exact colour of a dye is highly dependent on many other features of the molecule including molecular size and other substituents on the ring. bromoacid XL. Most auxochromes alter the colour of the dye slightly (hence their name). For example. NH2 N N N N NH2 SO3Na Figure 6. But if you order trypan blue you could get a bottle called chlorazol blue.3′-((biphenyl)-4. but the term light green is also used by some manufacturers for some basic .4′diylbis(azo))-bis(4-amino-1-naphthalenesulphonic acid) disodium salt. you tend to think there has been a mistake. so most histologists stick to using these. they simply alter the shade of colour. More of a problem is the fact that different dyes can be sold by different manufacturers under the same name. bromoacid S. whilst its common name is Congo red. If you ask for a dye by one name and get a bottle back with a different name on the label. Dye manufacturers usually give the dyes they produce common names such as eosin or Congo red rather than their full chemical name and some of these names are copyrighted. bromoacid XX. Use of common dye names Dyes are produced mainly for industrial uses such as textile dyeing.8 Structure of Congo red SO3Na Different manufacturers may have different names for the same compound and this can be very confusing.
g.6. When naming a dye in the description of a techniques. The common dye names are derived from their industrial use rather than their histological use. This is a monumental work of reference produced by The Society of Dyers and Colourists and each dye is given an individual number and listed along with its name(s) and properties. e. a few of the more important groups are listed below with examples of histological dyes from the group. e. In industrial terms.g. CI numbers are arranged according to their structure. Since each dye on the list has a unique number to identify it. e. an acid dye is one that would be used from an acidic solution and not necessarily one that would be anionic. all nitroso dyes have numbers between 10000 and 10299. methyl violet. e. with the most important feature being their chromophoric group. The quininoid ring is shown as the one on the left in the diagram below. eosin Y (CI 45380). with CI numbers up to 78000. a few examples are fuchsins. but since all three benzene rings are equivalent there can be rearrangement of the bonds and any of the benzene rings could take up this arrangement.g. martius yellow. The Colour Index To overcome all of the confusion there is a standard list of all dyes. the CI number should be given to avoid ambiguity. basic fuchsin is a basic dye.2 Dye structure 77 dyes that will stain the nucleus and not the connective tissues. neutral red is a basic dye in histological terminology. Sometimes the names coincide with histological properties. this list is the most reliable way of identifying a dye. picric acid. e. Azo dyes. Nitro dyes. These include the quininoid arrangement as the actual chromophore. Not all of these groups include important histological dyes. monoazo dyes have numbers between 11000 and 19999. There are 31 groups in all. C .g. fast green FCF (for Colouring Food) and brown FK (for Kippers). These have the -N=N. nitro dyes have numbers between 10300 and 10999. Triaryl methane dyes. There are a large number of dyes used in histology that fall into this category. their synonyms and their structures. methyl blue and aniline blue.g. This is called the Colour Index (CI).g. and so on. orange G.group (azo) as the chromophore. e. For example. These have the nitro group -NO2 as the chromophore. Buying the wrong dye can totally alter the results of a staining method. but they are sometimes misleading.
This group contains many important metachromatic dyes. whilst alizarin is most commonly used to detect calcium in tissues. methylene blue and azure A. Carmine is an important nuclear stain. but the middle ring now has S and N as constituent atoms. Examples include eosin and xanthene.78 Chapter 6 Staining theory Anthraquinone. Here the quininoid ring is seen as the middle of the three fused rings. For example. Most are used as nuclear stains and staining of cytoplasmic carboxyl groups is deliberately suppressed by using a slightly acid pH. Basic dyes are cationic and will stain anionic or acidic materials such as carboxylates. Two dyes within the same chemical group may have quite different uses in histology. such as toluidine blue. . since the actual binding relies on many properties and not just the simple ionic nature. Examples are alizarin and carmine. sulphates (many complex carbohydrates are sulphated) and phosphates (particularly the phosphates in nucleic acids). O Thiazine. This is very similar to the previous example in overall structure. Also. Xanthene. S N Histological classification In histology it is often more useful to classify dyes by their action on tissues and hence their uses in histology. The histological classification is only a broad guide to how a dye will work in practice. Acidic substances that stain with basic dyes are termed basophilic. Here the quininoid ring is the right hand one of the three fused rings and the ring is tilted compared with the previous example. dyes that are from totally different groups may quite easily be exchanged in histological techniques. the two anthraquinone dyes in the list above are used quite differently.
However. but these are on the same ion. Natural dyes are simply dye substances extracted from natural sources. and both of these properties are critical in the dyeing of textiles. most staining solutions contain other components to improve the staining. Neutral dyes are simply compounds of basic and acid dyes. The importation of indigo was strongly resisted by the dyers in Britain and the last woad mills closed only in the 1930s in Lincolnshire. Romanowsky stains are neutral dyes made from more complex mixtures. gallic acids from tree galls and ammonia from urine. Fast in this sense does not mean rapid but resistant to washing out or fading. Such dyes can stain either the nucleus or the cytoplasm if conditions are appropriate.3 NON-DYE CONSTITUENTS OF STAINING SOLUTIONS As well as dyes. Such dye complexes will stain both nucleus and cytoplasm from a single dye bath. carmine. Amphoteric dyes also have both anionic and cationic groups. orcein and litmus. with the mordant involved being able to mediate a dye–tissue interaction. Most are used to stain proteins in the cytoplasm and connective tissues. which are usually more reliable. Although the main source of dyes for early microscopists. Leishman and Wright’s stains. The dye woad was extracted from the leaves of Isatis tinctoria and is identical to indigo. they have largely been replaced by synthetic dyes. Organic materials were also used including tannins from wood bark. so their transfer to histology is hardly surprising. . Mordants Mordanting is the use of a non-dyeing compound to improve the binding of the dye. They are less common in histology but still very useful and include Giemsa. Substances that stain with acid dyes are called acidophilic. Alums were commonly used as mordants.6. These are the commonest dyes used in haematology. Natural dyes still in use include haematoxylin. the term mordant was very vague in its original usage and covered a number Box 6. This was despite woad processing having the reputation of being the smelliest of industrial processes. although synthetic varieties are also available for some of these. Mordanting of dyes has a long history and was crucial in early textile dyeing to fix the stain to the fabric and make it into a fast dye. 6. In this case.3 Non-dye constituents of staining solutions 79 Acidic dyes are anionic and will stain cationic or basic groups in tissues such as amino groups.5 Traditional mordants for textile dyes The methods used to make dyes stable and resistant to washing were often quite elaborate. cheaper and can be supplied more readily. both ions are coloured.
azo and nitro groups). The groups on the dye forming the dative bonds are mainly oxygen-containing (e. Regressive use of mordanted dyes It is also common to use mordanted dyes regressively (see Box 6. 6. hydrochloric acid. the selectivity of the dye is controlled by selecting the mordant not the dye.6). A self-differentiating haematoxylin .g. Since it is the mordant that binds to the tissue. The dye can only bind strongly to the tissue when the mordant acts as a link between the two of mechanisms of binding dyes. The mordant gives greater stability to the stain and is not easily removed by water. The excess mordant acts by displacing the dye lake and replacing it with a mordant with no attached dye.g. Staining is commonly done with the dye and mordant present in the same solution.g.9). The dye and mordant can also be used in two separate steps (e. Harris’s haematoxylin and carmalum).9 Mordanting.g. The term has been adopted for some histological staining. Theoretically it should be possible to devise a stain in which the balance of mordant to dye gives self-differentiation. The link to the dye would involve more than one such dative bond resulting in a chelate that was stable. thus forming the dye lake in the stain before being applied to the tissue (e. in phenols. but its use in histology is more restricted. Heidenhain’s haematoxylin) and one or two techniques have used post-mordanting in which the dye is applied first and the mordant added afterwards. the iron alum in Heidenhain’s haematoxylin can be used to slowly remove the excess haematoxylin. for example.e. as the stain resists decolorization by the later reagents. It is usually only applied to conditions where the mordant acts as a link between the dye and the tissue and where the mordant is a metal salt (see Fig. it is a fast dye) and this makes it ideal when other stains are to be used afterwards. The differentiation is done by using strong acids (e. carboxyls and quinones) or nitrogen-containing (in amine. alcohols or weak acids (i. The mechanism by which the mordant binds to the tissue is not certain but one likely mechanism is a dative covalency. Differentiation can also be done using excess mordant. often in alcoholic solution). The dye and mordant complex is sometimes called a dye lake.80 Chapter 6 Staining theory T I S S U E M O R D A N T DYE Figure 6.
By removing some dye. Regressive staining is often a better method of using stains. whilst the usual blue staining is called orthochromasia. their mechanism of enhancement is not known. potassium hydroxide in Löffler’s methylene blue and phenol in carbol fuchsin. The reason is that dyes are rarely specific and will not only stain the structure being demonstrated but will also slightly colour the background. since the background binding is usually weaker so the dye will be removed more readily. differentiation will simply bring the colour down to the optimal level. Accelerators are found in neurological techniques and are often hypnotic drugs such as barbiturates or chloral hydrate. Progressive staining is the simplest. As long as the object being viewed has more dye than is required. The large precipitate is more difficult to remove. 6. Trapping agents These differ from mordants in that they are always applied after the dye.4 Metachromatic dyes and metachromasia 81 Box 6. Accentuators are generally simply used to control pH. This colour shift that occurs with mast cells is called metachromasia.6. For example. however.4 METACHROMATIC DYES AND METACHROMASIA The term metachromasia is used when a dye stains a tissue component a different colour to the dye solution. Many dyes can show metachromasia but the thiazine group dyes are especially good for this type of staining. The removal of the excess dye is termed differentiation. whilst it can be removed from the more permeable Gram-negative organisms. Regressive staining involves overstaining the tissue so it is darker than is needed and then removing the excess to bring the colour down to the required level. The bestknown example is the use of iodine to trap the violet dye inside the relatively impermeable wall of Gram-positive bacteria. They form large aggregates with the dye and result in the dye precipitating in the tissue. it will also stain mast cell granules a pink colour. Accentuators and accelerators Accentuators and accelerators are materials added to staining solutions to improve the staining reaction. toluidine blue is a strong basic blue dye that stains nuclei a deep blue colour.g. One distinction is between progressive and regressive staining.6 Progressive and regressive staining The way in which dyes are used can differ. . the background can be cleared. with the dye being applied to the section until the desired density of colour is reached. which is generally not available since most mordanting is done with natural dyes that vary in their composition from one year to the next. was described by Baker in 1962 but requires a constant and reliable dye. e. albeit less than the required structure.
1 and Fig. The tissue on the left would be metachromatic as the dye has formed a polymeric form. This means that the colour absorption shifts to shorter wavelengths. Substances that can be stained in this metachromatic way are called chromotropes and they include mucins. The right-hand tissue would be orthochromatic as the dye molecules are widely spaced . Metachromasia requires water between the dye molecules to form the polymers and does not usually survive dehydration and clearing. 6. This is believed to represent polymerization of the dye. especially the sulphated mucins. The difference is in the spacing of the acid groups. Mechanism of colour shift in metachromasia The colour shift is always from a blue or violet dye to yellow or red staining. the middle tissue would be weakly metachromatic as the polymeric forms are only a few molecules in size.1 Target Staining theory Metachromasia and the spacing of acidic groups Distance apart of acidic groups (nm) 1.03 0. toluidine blue will stain hyaluronic acid a blue colour. The greater the degree of polymerization. leaving only the longer wavelengths to be seen.10.82 Chapter 6 Table 6. the stronger the metachromasia. For example.4 Staining Hyaluronic acid Pectic acid Mast cell granules Blue (orthochromatic) Blue/purple (weakly metachromatic) Red (strongly metachromatic) Metachromasia is important as it is highly selective and only certain tissue structures can stain metachromatically. pectic acid (found in plants) a purple colour and mast cell granules a definite red colour.10 Metachromasia. T I S S U E Dye Dye Dye Dye Dye Dye Dye Dye Dye T I S S U E Dye Dye Dye Dye Dye Dye Dye T I S S U E Dye Dye Dye Dye Figure 6. as shown in Table 6.5 <0.
Haematoxylin is soluble in both water and alcohol but dissolves faster in alcohol. haematoxylin itself is not a dye and for staining it must first be oxidized to haematein. OH O HO OH HO OH Thus. Haematein has two fewer hydrogen atoms and the rearrangement of bonds introduces the quininoid ring structure and hence colour. so stock solutions are often made by first dissolving the powder in alcohol and then diluting the alcoholic solution. which was named after the Campeche state in Mexico where it was originally found. since a counterstain should be a different colour to the main technique so that it does not confuse identification.5 Examples of important dyes and their uses in histology stains 83 6. for counterstaining this may not be the best stain to use. a blue haematoxylin stain is ideal. It is now cultivated in the West Indies. so other nuclear counterstains are needed. Haematoxylin Haematoxylin is a natural product extracted from the heartwood of the tree Haematoxylum campechianum. The formula of haematoxylin is shown below and it can be seen that there is no obvious chromophore and a solution of haematoxylin is not highly coloured. The logwood of the tree is first extracted with hot water and the dye is then purified by precipitation with urea. as can be seen from the formula below: OH O O HO OH HO .6. The dry powder is usually quite pure (about 95%) but is not actually a dye. However. The blue/purple colours of haematoxylin often overlap with the colour of the main technique. It is also easier to recognize tissue structure when only the nuclei are stained rather than when other structures are stained but the nucleus is unstained. since it is easier to recognize the location of the material in the tissue if a nuclear stain is used. Nuclear stains are important not only for looking at nuclear structure but also as counterstains for many staining techniques.5 EXAMPLES OF IMPORTANT DYES AND THEIR USES IN HISTOLOGY STAINS Nuclear stains Nuclear stains are very important in histology as the structure of the nucleus is often altered in disease. For simple nuclear structure.
since it may take months to prepare a new batch. which is a weak acid dye but has no mordant dye capability: OH OH O HO OH HO Most working solutions do not completely oxidize the haematoxylin and the unoxidized part gradually oxidizes to haematein at the same time as some of the haematein oxidizes to oxyhaematein. imparting a yellowish colour to the tissues. This ripening is considered to give a longer shelf life but it is inconvenient if supplies run out. but when combined with a suitable mordant. It is from this dye that the country of Brazil took its name. It can be used in the same way as haematoxylin but has never become as popular in histology. During the natural oxidation. The oxidation of haematoxylin to haematein can occur in atmospheric oxygen. many haematoxylin solutions produce precipitates that must be removed by filtration before using the solution.7 Brazilin Brazilin. a process called ripening. extracted from trees of the genus Caesalpinia.84 Chapter 6 Staining theory Haematein is less soluble in water and alcohol than haematoxylin. This is a slow process and can take months. potassium permanganate Box 6. Oxidation is slower in acid conditions. eventually all solutions will lose their strength and become useless. but is soluble in ethylene glycol and glycerol. Haematein is only a weak acid dye. is very similar to haematoxylin (only one OH different) and can be oxidized to brazilein. especially in cold and dark conditions. so that ripening is slower in the cold dark winter months than in the bright warm summer sunshine. OH O OH O OH OH O OH HO HO Brazilin Brazilein . so many solutions are deliberately kept acidic. haematein becomes probably the most widely used nuclear dye. However. Haematein itself can be oxidized further to oxyhaematein. Oxidation can also be carried out using oxidizing agents such as sodium iodate (200 mg per gram of haematoxylin). This replenishes the working solution and greatly lengthens the life of the reagent.
Carmine and carminic acid Carmine is a natural dye extracted from the red pigment cochineal. There is no need to ‘blue’ the sections in an alkaline solution since the mordant produces an intense black colour regardless of the pH. the alum dye lakes are quite soluble and have a strong red colour. Weigert’s). The use of iron haematoxylins has declined following the introduction of the celestin blue–haemalum sequence. although using smaller amounts than these traditional quantities will prolong the shelf life as explained above. which also resists acid decoloration. The ferric salts used are either ferric chloride or ferric ammonium sulphate (iron alum). e. Typical formulations include Harris’s. The resulting stain is blacker and more intense. Ehrlich’s and Gill’s haematoxylins.5 Examples of important dyes and their uses in histology stains 85 (177 mg per gram of haematoxylin) or mercuric oxide (500 mg per gram of haematoxylin. Other mordants that can be used to selectively stain specific tissue components are shown in Table 6. which is used in cooking. The mordant is therefore either used separately (Heidenhain’s) or the mordant and haematoxylin are mixed just before use (e. The mordant is usually either aluminium potassium sulphate (potash alum) or aluminium ammonium sulphate (ammonium alum). which may result in overoxidation and loss of staining. The ferric salts are oxidizing agents and will accelerate oxidation of the haematoxylin to haematein. These are the commonest haematoxylin solutions and there are many different formulae but they all have similar results. these staining solutions are referred to as haemalum solutions. Ferric salts: iron haematoxylin. Mordants for haematoxylin Haematoxylin is a very versatile stain and can be used to demonstrate many different tissue components in a highly selective way. . If the water is soft. calcium and other ions. Commercial carmine powder is quite variable in its composition and in addition to the dye–aluminium complex also contains protein. In alkaline conditions. Aluminium salts: haemalum. the dye lakes are less soluble and have a strong blue colour. In acid solutions. In hard water areas. The dyeing bath is usually acidified and once staining is complete the section is rinsed in an alkaline solution. Because of their use of alum salts as mordants. The haemalums are used regressively with a controlled differentiation in acid alcohol (1% HCl in 70% alcohol) followed by reblueing in water.6. lithium carbonate or tap-water substitutes.g. Mayer’s. the tap water is alkaline and simply rinsing in tap water will ‘blue’ the section.2. The type of mordant used alters the specificity and colour of the stain. Differentiation is often done with excess mordant and requires microscopic control. Carmine is actually a complex of aluminium and carminic acid rather than just the dye molecule. then an alkaline solution can be prepared. and will resist acidic counterstains such as van Gieson’s better than haemalum.g. Cochineal itself is extracted from the bodies of the scale insect Dactylopius coccus.
The loss of popularity occurred at the same time as laboratories switched to the use of modern synthetic mounting media. For many years carmine was a major stain with its main advantage being its permanence when compared with other dyes. The unreliability of the dye supply and the rising cost of the natural product have led to it becoming much less popular. the acidity of the mountant caused significant fading in just a few months.2 Staining theory Other mordants that can be used with haematoxylin Mordant Tissue element demonstrated Nuclei Myelin Elastic fibres Collagen Neuroglia Axis cylinders Mucin Fibrin Mitochondria Heavy metals (Pb.86 Chapter 6 Table 6. carmine was used for many different techniques and a great many methods were devised using carmine. as overstaining is difficult. it is better if the carmine solution is prepared from purified carminic acid. At the height of its popularity. when it forms the carmine complex. Any excess dye can easily be removed with 1% HCl. For precise staining. The problem with many other stains was that when they were mounted in Canada balsam. which cause much less fading of other dyes so there was less need for a stable nuclear stain. Carminic acid is the pure dye and is only slightly soluble in water but dissolves much better in solutions of an aluminium salt. Cu) and Ca Al or Fe Cr or Cu Fe Mo W Pb Al W Fe None (the metal in the tissue acts as the mordant and binds the dye) Carminic acid is a glycoside with a glucose derivative joined to an anthraquinone structure (see below). Solutions of the complex are not stable and significant deterioration occurs after only a few weeks of storage. H2COOH H H OH OH H OH HO OH O OH O H OH O CH3 COOH . A good carmine stain is easy for inexperienced workers to use.
Methylene blue was a major component of Romanowsky stains used in the staining of blood smears and bone marrow specimens. Methylene blue This is a very widely used simple blue stain that does not require a mordant. Cytoplasmic stains Cytoplasmic stains are often used as counterstains but can also be important to identify tissue components. it was used as ‘polychrome methylene blue’ (see Box 6. Many dyes will allow microorganisms to grow in them and this changes their staining characteristics. It gives a quick and simple nuclear counterstain for red primary stains.8–7.g. Both dyes are easily soluble in water and alcohol.6. It is often contaminated with methyl violet. Modern Romanowsky stains generally use mixtures of pure dyes rather than the empirical polychromed methylene blue. Most techniques are also used to distinguish connective tissue fibres and other protein materials. The cytoplasmic stains should produce several different shades of colour so that the tissues can easily .8 Polychrome methylene blue One of the methods used to produce polychromed methylene blue was to allow fungi to grow in a solution of it. Box 6. Neutral red and safranine These are popular red nuclear stains mainly used as counterstains to blue staining methods such as Perl’s iron staining method. Methyl green This blue/green nuclear stain is a useful nuclear counterstain and is also an important part of many techniques that differentiate between DNA and RNA in tissues.0 (turning yellowish in alkali). Neutral red can also be used as a vital stain when used at a very dilute (10–5) concentration.8).5 Examples of important dyes and their uses in histology stains 87 The most popular stains were the carmalum techniques (e. Grenacher’s) and acetocarmine. Mayer’s. The relationship between dyes and micro-organisms can be quite interesting. Neutral red can act as an indicator. In the preparation of the Romanowsky dyes. Other dyes have been used as antibacterial agents and the selectivity of dyes led many people to believe that dyes might prove to be the ‘magic bullets’ that would kill selected bacteria and save humanity from infection. The metabolic actions of the fungi converted some of the dye to other compounds. It is readily soluble in water and alcohol. Neutral red stains nuclei red and cytoplasm pale yellow. changing colour at pH 6. The polychroming produces a range of dyes from the original methylene blue of which azure B is probably the most important. but this can be removed by washing with chloroform.
although I have always found it difficult to distinguish more than about four. It is used when eosin staining is needed from alcoholic solution. This growth can be inhibited by adding a small amount of thymol to the solution and this acidic material also enhances the staining. The range of shades can be extended even further if more than one dye is used in the solution. Eosin is a very good cytoplasmic stain as it gives several shades to the tissue. Its pre-eminent role in staining is shown by the fact that many structures are referred to as eosinophilic when they will stain equally well with other acid dyes. although the parent dye fluorescein is widely used as a labelling compound in immunofluorescence. The dyes are also fluorescent but are solely used as red dyes.g. Eosin is also an important component of Romanowsky stains. which are all eosinates of azure dyes. when they will develop significant growth. . eosin Y (yellowish) changes to eosin B (bluish) if the bromine groups on positions 2′ and 7′ are changed to nitro groups. All are derived from fluorescein. However. It must be differentiated in alcohol. Fast green FCF and light green SF These are green anionic dyes similar to the blue dyes above and are frequently used as counterstains to red dyes. Both are quite large dye structures and are frequently used to stain connective tissue fibres. Ethyl eosin is an ester rather than the more usual sodium salt and is only slightly soluble in water. They are often confused as both are also known as soluble blue and water blue. adding dilute acids will improve eosin staining but may overdifferentiate the nuclear stain. Eosin solutions keep reasonably well unless they become contaminated by fungi.88 Chapter 6 Staining theory be distinguished. Some workers claim that up to seven different shades can be distinguished. Eosin gives a good red cytoplasmic counterstain but if other colours are required then other dyes must be used. The sodium salts of the dyes are all freely soluble in water and fairly soluble in alcohol but will precipitate as eosinic acid if the pH is very low. a variety of shades of red can be produced from yellowish to bluish e. Both are water soluble but insoluble in alcohol. By substituting halogens or nitro groups for some hydrogens. Eosin This is not a single dye but a variety of related dyes. which is a useful fluorescent dye widely used to label antibodies but is useless for ordinary light microscopy. Methyl blue and aniline blue These are widely used blue anionic dyes with similar staining properties. Most of the stains are acidic (anionic) dyes but can be used in mixtures to improve the contrast between different components. Fast green FCF is less prone to fading than light green SF.
The red blood cells should thus be stained with the smallest dye. Orange G is soluble in water but less so in alcohol and is a major component of the Papanicolaou stain used in cervical cytology. when two acid dyes compete for binding to tissue. would stain all cytoplasm and connective tissue. The larger dyes will also be able to form more van der Waals forces. In addition. Picric acid is a valuable stain in multiple acid dye techniques because of its small size. Van Gieson’s stain This stain uses two acid dyes (acid fuschin and picric acid) to distinguish between acidophilic materials. stains red. metanil yellow and martius yellow These are very pale-coloured dyes ideal for faint background staining or in conjunction with other acid dyes. Most other cells types. picric acid (trinitrophenol). whilst muscle and erythrocytes. . the larger dye will generally tend to displace the smaller dye. which restrict access of larger dyes. In the less-dense collagenous tissue. whilst erythrocytes are packed full of haemoglobin and are much denser. Acid dye combinations The differing molecular weights and sizes of dyes affect their diffusion rate and their ability to permeate into small spaces in the tissue. By combining them in a single solution. the tissue differences can be exploited. The main way of distinguishing the fibres and cells is by using a combination of acid dyes to stain different structures in differing colours. the large and small dyes will be in competition. The larger dye will dominate and the collagen will appear stained only with the largest dye. Tissue permeability is related to the amount of protein that is present and the amount of water between the proteins. the smaller dyes tend to be paler colours (yellowish). Each dye. allowing free and rapid access to both dyes. if used alone. Thus. These effects combine so that smaller paler dyes are overwhelmed by larger denser dyes when they compete directly. Loose collagenous (areolar) tissue has many minute fluid spaces and is very permeable. Both dyes are mixed into a single solution along with hydrochloric acid to give a pH of 1–2. The concept of the differential acid dyeing technique is that only the small dye will penetrate into the dense red cells. stain yellow. Connective tissue methods Connective tissue consists mainly of collagen fibres. The open texture of the collagen. elastic fibres.6. including muscle. whilst the larger dyes are dense colours.5 Examples of important dyes and their uses in histology stains 89 Orange G. glycosaminoglycans and cells. lie between these two. There is still uncertainty about the exact mechanisms of these techniques but they seem to depend on differences in dye size and differing permeabilities of tissues.
Trichromes also differ in that the three dyes are usually used separately and sequentially. Alcoholic solutions seem to affect penetration by allowing dyes to permeate more freely. only the largest dye of the competing dyes does the staining Heteropolyacids aid trichrome staining Trichromes differ from van Gieson’s stain in that an extra reagent is used in the form of one of the heteropolyacids.90 Chapter 6 Staining theory Permeability and dye size considerations would suggests that the small dye will rapidly penetrate both the dense red blood cells and the looser connective tissues. In each case. Molecular size and permeability: not the full story Although the explanation given here accounts nicely for much of the staining with multiple acid dyes (trichromes and van Gieson’s). acid fuchsin (FW 578) and methyl blue (FW 800) can be used as a trichrome mixture. if the dyes are used alone they will readily stain all of the tissue. showing that it is able to penetrate these structures. rather than in a single mixed reagent as in van Gieson’s stain. This makes the molecules act as if they were a slightly smaller size. the fuchsin will displace or mask the paler picric acid with the result that the connective tissue will stain red but in the red blood cells the picric acid will not be displaced or masked and the red cells will stain yellow (see Colour plate 6). the fuchsin dye in van Gieson’s stain will stain red blood cells. the intermediate cytoplasm and muscle cells are stained red by the intermediate-sized dye and the collagenous tissue stains with the largest dye. In particular. The larger fuchsin molecules will penetrate into the connective tissues quite readily but will penetrate the denser red blood cells only slowly. For example. possibly by increasing the size of tissue spaces. Trichrome stains These take the differential staining a stage further and use three differentsized dyes to selectively stain the three tissue densities. picric acid (formula weight (FW) 229). Trichromes can to some extent be ‘tuned’ to differentiate between tissue fibres by selecting dyes of appropriate sizes and by controlling the size of tissue spaces. there are anomalies and the exact mechanisms are still very much undetermined. a red blood cell is less than 8 µm across at its widest point and less than 3 µm thick. The red blood cells are the densest tissue and stain with the smallest dye. These improve the staining but whether they simply act as colourless dyes or have a more active role in some form of mordanting is still unresolved. . The heteropolyacids are either phosphomolybdic (sometimes called molybdophosphoric) acid or phosphotungstic (tungstophosphoric) acid. Thus. Where both dyes are present. There is the possibility that the timing is crucial and that by using a limited time the red dye would not have long enough to penetrate into the cells. Even on theoretical grounds this seems unlikely. For diffusion across such small distances to take more than 2 min (which is a typical staining time for van Gieson’s stain) would suggest an extremely dense material.
otherwise the dye may be completely different. just better. One manufacturer. dextrans and even other dyes. which makes quality control in the histological laboratory difficult. Nobody thought his dyes were purer. Grübler dyes lost their leading role following the Second World War when importing of dyes from Germany became impossible and laboratories had to find other sources. some laboratory suppliers offer certified dyes that have been tested biologically for their stated uses. are often impure substances and may contain significant amounts of other materials such as salts. The situation is more complex than the simple dye size and permeability would suggest.6. Some older samples of dye certainly seemed to have an insoluble residue left after preparing the staining solution. it is worth checking that you have not got a different batch of dye to the usual one. If acid fuchsin is used in combination with other dyes. The non-dye constituents are often very important and may grossly affect the staining. Dye manufacturers therefore adjust their products to give consistent dyeing of fabrics rather than histological reliability. it will stain erythrocytes if the other dye is larger. but the same dye can stain both under different conditions. It is always the smallest dye that stains the red blood cells and the largest that stains the collagen. yet the concept does seem to hold in most practical applications and several good trichrome methods have been produced on the basis of this theory. there is no real major difference in the result. however. It was said that ‘not only does Herr Grübler have the best dyes. This means that dyes.5 Examples of important dyes and their uses in histology stains 91 If van Gieson’s staining is extended to 30 min or more. unlike most biochemical reagents. but will stain collagen and not erythrocytes if the other dye is smaller. The important property for textiles is a reliable final colour rather than chemical purity.8 Contaminated dyes Dyes have always been impure so it became important to have a good source of dyes. Dyestuffs are quite expensive to manufacture. It is also worth repeating that some dyes have many names and it should always be made clear which dye is needed by using CI numbers. became famous for the quality of his stains and if you read the old textbooks you will find his dyes being recommended time after time in techniques as being the best available. Different batches of dye will differ in their dye and contaminant content. so it was not unknown for dyes to be ‘cut’ with less expensive materials to make them more profitable. There is a growing tendency for laboratories to buy in many reagents in a ready-prepared form rather than making up stains from the original Box 6. . he also has the best impurities’. Dyes and quality control As mentioned earlier most dyes are not produced for histologists but for textile dyers. To try to combat this problem. The actual content of the named dye rarely exceeds 95% and may be as little as 25% of the total weight. Such dyes are more expensive but should match their stated uses reliably. When a staining method suddenly stops staining as expected. Similar anomalies can also be seen when different combinations of dyes are used.
fuchsin samples may be good for use in Ziehl–Neelsen staining for mycobacterium but not for preparing Schiff ’s reagent. Advantages Silver impregnation has a number of advantages compared with dyeing techniques and has a number of very common applications. Measurement of absorption (including a full spectrum if a suitable spectrophotometer is available) can be used to determine the amount of dye in a sample and may also show contaminants. refrigeration) will usually give details. black. as many of the techniques used in quality control require complex equipment to analyse the dye samples (e. 3. infrared spectroscopy. This will detect coloured contaminants of dyes and can be a sensitive way of comparing two dye batches. but the mechanism is quite different to the effects of dyes and the structures are actually plated with the silver rather than the silver being reversibly bound to the section. Once the dye has been made up into a solution it may not be permanently stable. for example. Most techniques using reagents that need special storage (e. although the dark glass will also mask any contamination and precipitation. Dyes can alter due to oxidation by the air.6 SILVER IMPREGNATION Metallic impregnation is an alternative way of increasing the contrast in tissues. then storage of the reagent in brown bottles to prevent light reaching the dye may help. contamination by micro-organisms growing in the solution or chemical reactions between constituents of the dye solutions. high-performance liquid chromatography). This leads to more consistency in the laboratory as the scale of industrial production can be controlled more carefully than small irregularly prepared batches in the laboratory. fine deposit of silver and silver oxide where the silver ions have been reduced. which produces a dense. Some dye batches may be suitable for one stain but not for others. The main advantages of silver techniques are: . The commonest metal to use in light microscopy is silver. Silver impregnation is also called silver staining. so care must still be taken.g. bleaching by light. Simple paper chromatography using filter paper is often enough to pick out impure dye samples. Reagent bottles should be clearly labelled with the date of preparation and renewed at regular intervals or sooner if the staining seems to be suffering.g. Checking dyes in a histology laboratory Quality control of dyes within the laboratory is difficult.92 Chapter 6 Staining theory ingredients. If light accelerates the deterioration. but some simple tests can usually be performed. 1. Testing with standard dyeing techniques to determine whether the dye is suitable or needs altered staining times/conditions. 6. Chromatography. 2.
which gives good contrast and is excellent for taking photographs.6 Silver impregnation 93 1. The silver deposit is densely black. Silver cannot be discarded into the drains as it is a heavy metal poison. This can be useful for fine fibres such as reticulin or for slender bacteria such as spirochetes. This can extend to different workers. The end product is metallic silver. 7. Strong alkaline solutions have a tendency to strip sections off the glass slides so extra care and adhesives are needed. 3. These materials include reticulin fibres (see Colour plate 7). Silver techniques are very sensitive methods and will detect many materials that are difficult to demonstrate by dyeing. 2. The techniques can be unreliable and capricious. The silver solutions are often very alkaline. Disadvantages 1. 3. 4. e. Silver is expensive. They will sometimes work well and other times will not work at all. Silver is very difficult to remove without using dangerous reagents. even when using the same reagents. 5. but as they look just like water it is not always obvious that there has been a spillage.6. Dyed sections rarely last more than 10 years without some signs of fading. Silver solutions are easy to wash out if they are caught early enough. Once reduced. laboratory coats. so clothing is often permanently stained. 6. which if properly fixed and washed is effectively permanent. which is then soluble in sodium thiosulphate solutions.). They are stable and do not fade. which are difficult to observe with haematoxylin and eosin staining but can be readily demonstrated with silver impregnation. 2. which converts the silver to silver iodide. whilst everyone else struggles. etc. glassware. Some silver solutions have a tendency to become explosive if stored for more than 24 h. There sometimes seems to be one person in the laboratory who can get a technique to work perfectly. 4. Silver techniques are so sensitive that they can sometimes give nonspecific background deposits (‘dirty preparations’). motor end plates and astroglia (see Colour plate 8). Slender objects are thickened because they become silver-plated. . benches. the safest way to remove silver deposits is by using an iodine solution. The silver deposit in black and white photographs is similar to the material produced by silver impregnation and photographs from 150 years ago are still in excellent condition. Metal impregnation methods are more common in neurological methods. The techniques have a tendency to stain everything they come into contact with (hands.g. Staining times can vary tremendously from one day to the next when a fresh batch of silver solution is prepared. for axons.
atoms are deposited in this initial stage and these are too small to be visible. This autocatalytic activity makes silver useful in many fields other than histology. The initial reduction reaction with silver only deposits submicroscopic atoms of silver at particularly reactive sites. perhaps as few as two. even with high-power microscopy. The strong reaction in this case is due to phenolic components (5-hydroxytryptamine. or serotonin).11). These silver atoms are deposited at the site of reduction.94 Chapter 6 Staining theory Use of silver Silver is not the only metal that can be used for impregnations but is the most useful as it is easily reduced and any reduced silver acts as a catalyst for the reduction of more silver. e. The section is treated with silver solution (silver nitrate) and the phosphates and carbonates in the mineralized bone form insoluble silver salts. 6.g. the argyrophil reaction and ion-exchange reactions. Ion-exchange reactions Ion exchange can also deposit silver and this is used to detect mineralization of bone using the von Kossa technique. Probably only a few. which derives its alternative name (argentaffin pigment) from the reaction. possibly by ionic mechanisms as for dyeing. Silver solutions are reduced during the impregnation. This type of reaction where an external reducer or developer is added is called an argyrophilic reaction. The reaction only needs the addition of the silver solution. These silver atoms then act as catalytic sites where more silver can be deposited by the reducing action of a developer (see Fig. formaldehyde or hydroquinone (quinol). These are the argentaffin reaction. so silver techniques are primarily methods for reducing materials. the tissue contains reducing groups that are sufficiently strong and present in sufficient quantity to give a visible deposit without added reducing agents. The silver salts are then blackened by . There are three different ways of producing silver deposits. The silver is mainly adsorbed as silver ions but small amounts are reduced to silver atoms. The argentaffin reaction occurs particularly with reducing pigments and is strongest with the pigment of enterochromaffin cells. The use of silver is widespread in photography and the chemistry of photography and the chemistry of silver impregnation are very closely related. but tends to be very slow and may take up to 24 h to give a deposit. such as in the Masson–Fontana technique. The argentaffin reaction In the argentaffin reaction. In this case the developer does the main reduction and the tissue simply provides places where there are silver atoms to catalyse the reduction. These groups are often aldehyde groups and silver solutions can be used to replace the Schiff ’s reagent in the periodic acid–Schiff technique (see Chapter 7) to give periodic acid–silver. The argyrophil reaction Many tissue groups are able to adsorb silver.
6. Silver nitrate This is the commonest form of silver salt used in the preparation of silver solutions. If they are being used in a glass container. Several silver solutions can be used in silver techniques but they are not directly interchangeable as they differ in their sensitivity to reduction. the method actually detects carbonates and phosphates.g. Silver solutions always need careful preparation and some diamine silver solutions can become explosive if kept for more than 24 h. if an explosion occurs the glass fragments will be held by the sticky tape.g. e.6 Silver impregnation Silver ions Silver atom Ag 95 Ag Ag Ag Protein Protein with one reducing site HCHO Ag Ag soln Ag Ag Ag Protein Ag Ag Ag Ag Ag Ag Ag Ag Ag Ag Ag Ag Ag Protein Protein Catalytic reduction Figure 6. then a simple safety precaution is to wrap them up with adhesive tape (Sellotape). CaCO3 + 2AgNO3 → Ag2CO3 + Ca(NO3)2 Ag2CO3 (UV treated) → Ag2O + CO2 Black Silver solutions Ammoniacal silver solutions are used as they are easily reduced. . Simple silver nitrate solutions are sometimes used. or as sensitizer solutions. von Kossa’s solution. as tap water will react with the silver salt. Although often said to demonstrate calcification of bone.11 Silver deposition and reduction in the argyrophil reaction UV light or hydroquinone solutions (see Colour plate 7). e. It is important always to use distilled water in any silver method. but for most techniques a more readily reduced form is needed. Beilschowsky’s method for nerve fibres.
This involves using ‘gold chloride’ (sodium chloroaurate): 3Ag + (AuCl4)– → Au + 3AgCl + Cl– Thus. When mixed with silver nitrate. It is probably not necessary in histological preparations. but has the advantage of being very sensitive. Hexamine silver solutions These use hexamine (methenamine or hexamethylenetetramine). but it is always done ‘just in case’. previously called hyposulphate). Very small deposits can often be reduced by toning.96 Chapter 6 Staining theory Silver diamine Silver diamine solutions are prepared by precipitating the silver with a hydroxide solution and then redissolving in a minimum amount of ammonium hydroxide. The precipitate is then dissolved using strong ammonia as for the diamine solution. 2Ag+ + 2OH– → Ag2O + H2O Precipitation of silver oxide Ag2O + 4NH3 + H2O → 2[Ag(NH3)2]+ + 2OH– Dissolving to form silver diamine Silver carbonate Silver carbonate solutions are prepared by precipitating the silver using either lithium or sodium carbonate solution. Silver carbonate solutions are claimed to be even more sensitive than diamine solutions. this produces a white precipitate that immediately redissolves without the need to titrate with strong ammonia. . as all of the silver is usually completely reduced so there is little risk of further reduction. Gold toning also alters the colour from an intense black to a warmer brown/black colour. Background deposits Silver techniques often produce a non-specific deposit due to contaminants. so an adhesive is often advisable. This removal of the precipitating salt is different to the previous example of silver diamine where the hydroxide is left in the solution. Following completion of the technique. This is a photographic fixer that dissolves excess silver ions and prevents them later depositing as background. These solutions are very alkaline and this makes sections more liable to detach during staining. The final solution can be explosive if it is stored for more than 24 h. three silver atoms are replaced by one gold atom. The precipitate is filtered and washed. For very small deposits this will result in a great reduction in size (thus reducing the background staining) but the large deposits of the impregnated tissue will hardly be affected. the sections are usually treated with ‘hypo’ (sodium thiosulphate.
This wide variation might suggest that the technique is quite insensitive to conditions and would work reliably. ambient temperature and ambient light. water quality (both tap and distilled or deionized water). These variations include tissue fixation and processing. Silver techniques are more difficult to get exactly right than most staining methods and require care.6. Silver techniques vary quite widely in their conditions There are many variations on silver techniques that seem to give good results. First the reticulin is oxidized to give aldehyde groups: OH Reticulin OH KMnO4 Oxidation Reticulin CHO CHO Then the silver solution oxidizes the aldehydes to acids and in the process is itself reduced to silver atoms that precipitate at the site of reduction: CHO Reticulin CHO COOH Reticulin COOH 4Ag 8NH4 4OH 4[Ag(NO3)2 ] 3H2O Aldehydes are one of the commoner reducing groups in tissues and silver solutions can often be used to detect the presence of aldehydes. patience and experience to get an even impregnation and lack of non-specific background. The wide variation is actually a reflection of this. There are probably differences between the laboratories that are not particularly mentioned or even controlled that make one method more suitable for one laboratory than another. but this is not the case. It is largely a matter of preference which technique works best in a particular laboratory. regardless of any slight technical errors. The actual concentrations of silver vary quite markedly from 1 g per 100 ml (Foot method) to 10 g per 100 ml (Laidlaw method).6 Silver impregnation 97 Silver techniques Reticulin can be demonstrated using silver impregnation and the following is a fairly typical silver staining technique based on the method proposed by Laidlaw in 1929. Times and temperatures also vary from 30 s (Gordon and Sweet method) to 60 min (Perdrau method) and temperatures from room temperature of 20°C up to temperatures of 70°C (Lillie method). since many .
alcohol or xylene for quite long periods. Treatment with xylene for 5 min is usually sufficient. as this can introduce minute air bubbles into the protein mesh. 95%. The dry labelled slides are placed in the rack and the wax is removed. Removal of the wax needs to be complete. if any wax remains it will result in uneven staining. For this reason.8 Dewaxing or ‘taking to water’ If the section is frozen then the phrase ‘take the section to water’ can be ignored as the section is already in water. Section rehydration For staining. which is the cellular structure. This process of returning a paraffin section to water is usually called either dewaxing. The sections can then be transferred through a series of graded alcohols (typically 100%. 70%) and finally into distilled water. They do not need prolonged times in any of these baths since penetration is very rapid through the thin sections. This is conveniently done using stainless steel racks that hold a number of slides and flat staining dishes. Once rehydrated. 30 s with gentle agitation will usually be enough. It is also bad practice to allow sections to dry out at any stage in the staining. especially if kept in water. which can totally ruin the section. . There are occasionally instances when sections need to be dried. or ‘taking the section to water’ (see Box 6.7 GENERAL TREATMENT OF SECTIONS DURING STAINING After drying to achieve adhesion. although they do gradually lose adhesion. it is hardly surprising that it will also remove dried-on sections. personally I prefer the phrase ‘take the section to water’ to ‘dewaxing’. the wax must be removed and the section rehydrated. as it is less prescriptive and suggests that the histologist needs to get the section into water from the medium the section is currently embedded in. paraffin wax sections are still not ready to be stained. Since soaking will remove dried-on food. which forms a waterproof coating and prevents dye access to the proteins. One of these phrases will be found Box 6. they will slowly deteriorate. This is similar to the ‘soaking’ of pans with dried-on food residue. but there is no problem with shrinkage at this stage because the tissues are firmly attached to a rigid slide. Often students do not think and try to process the tissue as if it was a paraffin section. Xylene is still the most commonly used reagent for this process. to get an automatic and reliable technique. 6. Sections are best dewaxed immediately before being stained. as they are totally impregnated with wax. Deterioration is not rapid and sections can be kept in water. and largely failed. Xylene is less commonly used for processing tissues because of its tendency to cause shrinkage and hardening.8) or occasionally by the somewhat grander phrase of deceration. but these are the exception. and hardening is no longer a difficulty as there is no further sectioning to be done.98 Chapter 6 Staining theory people have tried. These air bubbles may remain and not be removed when the tissue is again placed into reagent and will end up in the final preparation.
an overstained section will give better results than the usual staining intensity. 1 min) so that sections can be added at any time and will follow the same path. The use of an absolutely regular procedure ensures that there is little variation in results. Machines are fine for doing hundreds of haematoxylin and eosin stains. regardless of their requirements. Automated staining also demands reproducible reagents. Most histologists can easily compensate for gradual changes in reagents as they age or for sudden alterations from a new batch of stain without too many problems. It also does not lend itself to situations where different results are needed. These machines are inefficient for staining single sections. This inflexibility may also result in reagents being discarded sooner than they would be for manual staining in order to maintain a standard program. It is also only feasible for techniques that are carried out for a large number of samples. Once fully rehydrated. An ordinary stain will give insufficient contrast for the film’s recording capabilities but a more-intense stain will give stronger differences between the tissue components. Thus. when photographing at low magnifications. Automated staining machines are also less flexible in producing single stains. these machines must go through the full cycle before another section can even begin since the steps are uneven. Machines only follow the program and cannot tell that there is any need to change. if a different batch of reagent is prepared.6. Automation frees staff from a routine task that is relatively straightforward and allows them to do more demanding tasks. but it is not reasonable to use a machine for stains where the technique is only required for two or three slides each day. producing a single slide may hold up some types of machine. so that direct comparisons are valid from one batch of stained sections to the next. These machines often cannot cope with large numbers of sections in a short space of time. for example. The disadvantage is that there is less flexibility. . Any alterations result in machines needing to be reprogrammed. Automated staining Automated processing of tissues is widely accepted and a similar automation is possible with staining.g. even when they are already programmed for that stain. if a longer time is needed. All of the sections will be given the same treatment. This accuracy and reproducibility are crucial in some applications such as diagnostic and exfoliative cytology (see Chapter 12) where the colour of the cytoplasm is an important diagnostic feature. An alternative strategy is to have all the steps the same length (e. The difficulty here is that. The same general principles apply to both situations. then several baths of the same reagent are required. the machine will not recognize this and compensate for the change in the way that a person would. for example. the sections can be stained in aqueous reagents until they are ready to be mounted. If there is a change in a reagent’s staining properties.7 General treatment of sections during staining 99 at the beginning of most staining schedules and must always be done when paraffin sections are used.
You only need to compare an unmounted wet section at the end of staining with a properly mounted section to see the difference. They have found a significant role in two main areas: 1. There is less requirement for variety in mounting.100 Chapter 6 Staining theory Automated staining machines are very useful for absolute regularity with large numbers of sections needing the same treatment at the same time. a section mounted in a high refractive medium has only two refractions. so automation is possible. The difference can be seen in Fig. 1 coverslip) for the best results. The coverslip should have a thickness of 0.12 where the optical paths through a wet section and a mounted section are compared. On the left.12 Effect of a coverslip on viewing sections. Here the actual numbers are smaller but the need for absolute consistency is greater.17 mm (No. The unmounted section has twice as many refracting surfaces and the opaque tissue will transmit much less light. 6. so provided they are working well these machines are a useful addition to the laboratory.8 SECTION MOUNTING Use of coverslips Mounting of sections under a coverslip is essential to get the best and clearest view of the specimen. whilst a section in water has up to four refracting surfaces . Haematoxylin and eosin staining in histology. as this is the thickness used in optical calculations. The process of mounting sections is very mundane. The microscope manufacturers usually assume that the specimen will be mounted in a medium with a high refractive index and covered with a thin glass coverslip and calculate all of their optical corrections on that basis. Immunohistochemistry. 6. so these techniques have moved to more automation. Papanicolaou staining in cytology and blood-film staining in haematology. Thicker Figure 6. nucleic acid hybridization and similar techniques. 2. This is because the sheer numbers needing staining make it worthwhile. The use of automatic coverslipping machines is often linked to automated staining.
There is no single mounting medium that is suitable for all specimens and stains. brittle. This was the original resinous mounting medium used in histology. yellow solid. Clarity under normal conditions of use.5. especially in the light. Some media can become opaque as they dry out and are not suitable for long-term preservation. Approximately 60 g in 100 ml of xylene gives a good working mountant. Most tissues have an RI of between 1. Some media may also act as solvents for the dyes and as a consequence the dye diffuses or leaches out into the mountant. If the RI is much lower than 1. This is usually a disadvantage as it reduces clarity but it can sometimes be an advantage as it will give some contrast to even unstained tissues. This will gradually obscure the tissues. Setting. 2 coverslip will interfere marginally with the clarity and very thick coverslips may even prevent the oil-immersion lens being used as they can have a greater thickness than the normal working distance of the oil-immersion lens. Some mounting media will cause fading. 5. There are two major types of mounting media used and the difference is in the solvent.5 and 1. Canada balsam is derived from the Abies balsamea fir tree and is available as a dried. The ability of a mountant to dry or set quickly and hold the coverslip in place is very useful. 2.8 Section mounting 101 coverslips such as a No. This is really only critical for fluorescence microscopy but it is generally a useful characteristic for a general mounting medium since it eliminates the need to use a special mountant when fluorescence is being used. Fluorescence.6. The commonest types are the resinous mounting media. which will cause significant fading. so a mounting medium with an RI in this range will give maximum clarity. This is most common with acidic mounting materials. The yellow colour of the mountant hardly seems to matter when viewed through . Refractive index. although it takes a few days to dissolve completely. if the dye or histochemical reaction product is soluble in xylene. 4. Many aqueous-based media fail to harden sufficiently and the coverslip will need ‘ringing’ to preserve the section. 3. usually xylene. Water-based mounting media will accept tissues straight from distilled water and are used when a xylenebased medium would not be appropriate. and which need the section to be dehydrated and cleared before mounting.g. Effects on the stain itself. Resinous mounting media Canada balsam. then tissues will not be completely transparent and diffraction will occur. which are based on hydrophobic organic solvents. Mounting media The mounting medium should have a high refractive index (RI).55. The properties that need to be considered in a mounting medium are: 1. e. It will melt at high temperature and is soluble in xylene.
Aqueous mounting media There is no fully satisfactory aqueous medium and several different ones are used for different purposes. It has an RI of around 1. It is relatively expensive and is mainly of historical importance rather than being a common mountant. This uses a gum (gum arabic or gum acacia) and sucrose to raise the RI.g. The addition of p-phenylenediamine is said to retard the fading of fluorescence. Glycerol jelly. it needs to be dehydrated and cleared in xylene before finally being mounted. It is solid at room temperature and needs to be melted in a waterbath before use. The initials come from the components: Distrene 80 (a commercial polystyrene). so it can give nicely transparent . especially of basic dyes. although it can be ringed for slightly longer use. for immunofluorescent antibody techniques. for example. Glycerol.g. The specimens do not need ringing. Other synthetic media are available such as Permount or Entellan. Sections may also allow the growth of organisms in storage. DPX. This is a synthetic polystyrene resin that is dissolved in xylene and has some plasticizer added. so it is best thought of as a temporary mount. The mountant is usually significantly acid and will cause fading.33) is raised sufficiently to give a clear image. They differ in the way in which the RI of water (1.102 Chapter 6 Staining theory the microscope. Tissues do not need any treatment before mounting and can be mounted directly from water or buffer. This is simply the reverse of the dewaxing. which are commercial brand names. which can interfere with the mounting medium. but it still does not keep well.42) than most mounting media. so there is usually an antibacterial additive (e. Apathy’s medium. Most are best considered as temporary mounts and need ringing to hold the coverslip in place and prevent drying out.5. It can be used alone or with the addition of a buffer to control the pH. but it is better not to use the same reagents as they gradually become contaminated with the other reagents. dewaxing will leave wax in the xylene. It is a useful medium for fluorescent staining. When a section comes to the end of normal staining and is ready for mounting in a resinous mounting medium. It is very easy to get air bubbles trapped in this medium. phenol). e. plasticizer (e.g. It neither hardens nor dries out and is usually used as a very short-term mountant. dibutyl phthalate) and xylene. It has very little tendency to fade dyes and hardens in about 24 h. so the clarity is reduced and some unstained structures will be visible. It is a water-white clear solution and is one of the more popular mountants in use. The usual formulation has a lower RI (1. Glycerol jelly is quite a good growth medium for some bacteria and fungi. so it is convenient to melt it and get rid of any air bubbles by warming it in a vacuum-embedding oven. Glycerol is a trihydric alcohol with a high RI. This uses the addition of gelatine (up to 12% in some formulations) to allow the medium to set.
They solidify slowly by evaporation but specimens can be ringed to prevent this. Paraffin wax can also be used. These are more permanent than the other water-based mounting media.6. Again the solvent is a theoretical problem but I have not had problems. SUGGESTED FURTHER READING Gamble. which makes it convenient and simple. but are still not as good as a resinous medium. They dissolve in water or buffer but need constant stirring.D. (2002) The hematoxylins and eosin. although I have never found this to be a problem. Provided the slide is dry. Stained slides should be stored away from light as the dyes will fade even in the best mountant. and Wilson. M. Edinburgh: Churchill Livingstone. The only drawback is that it is dissolved in acetone. . Polyvinyl alcohol or polyvinylpyrollidone media. Ringing was done on a turntable to give a nice neat finish. In Theory and Practice of Histological Techniques. Storage of slides Mounted slides should always be carefully labelled and stored horizontally until fully dry and set when they can be stored on their edge or end. It has a tendency to crystallize in storage and can set by drying but this is quite slow. Ringing was originally so called because the coverslips were round and so there was a ring of the sealant round the coverslip. Temporary mounts need ringing Ringing is the term used for sealing the edges of a coverslip when the mounting medium does not set. These are synthetic and less liable to bacterial contamination than the organic-based mountants. which immediately sets. A piece of warmed metal (such as the flat end of a broad spatula) is used to apply a layer of molten wax.8 Section mounting 103 preparations. Many styrene-based cements can also be used and again are convenient as they come in tubes ready to squeeze out around the coverslip. Originally it used a gold size followed by a black asphaltum varnish. Gamble). Good temporary ringing can be achieved in a number of ways. which may affect some materials. although the addition of phenol is still advisable. I. These cements can often be semi-permanent. Again. Bancroft and M. this is quick and easy but is easily broken and will not store well. Ordinary nail varnish works quite well and comes in a bottle with its own brush. Most laboratories have dropped this and ringing is now just a temporary expedient rather than an aesthetic requirement. This produced a very neat finish and some commercial suppliers of prepared slides still finish many of their preparations in a similar way as it looks good. 5th edn (eds J. it may need the addition of an antibacterial agent to help preserve it.
10. 3. How do pH and salt concentration alter dye binding? 4. Explain this oddity. Why might a lipid-staining technique recommend mounting in glycerol jelly instead of DPX? 13.D. In Theory and Practice of Histological Techniques.A. Bancroft and M. Bancroft and M. (2002) Connective tissues and stains. Gamble). Why is silver the best metal for metallic impregnation techniques? 12. 3rd edn. Name one red and one blue nuclear stain. (2001) Histological and Histochemical Methods.104 Chapter 6 Staining theory Horobin. M. 5th edn (eds J. In Theory and Practice of Histological Techniques. (2002) Theory of staining and its practical implications. Oxford: Hodder Arnold. Edinburgh: Churchill Livingstone. Why do some haematoxylin solutions initially improve with keeping and then deteriorate? 9. Edinburgh: Churchill Livingstone. 5. R. 11. Outline why permeability and dye size might explain trichrome staining with three acid dyes. Distinguish between argentaffin and argyrophil silver impregnation. Gamble). What is the role of chromophores and auxochromes in dye structure? 2. Why do most laboratories routinely use a resinous mounting medium? . What is the name of this phenomenon? Why does the colour change occur? 7. SELF-ASSESSMENT QUESTIONS 1. 5th edn (eds J.D. A small amount of mordant causes staining but an excess of mordant removes the staining. Lamar Jones. Why does haematoxylin mordanted with aluminium salts stain nuclei but other mordants cause haematoxylin to stain connective tissues or nerve fibres? 6.W. When would you use a red nuclear stain and when would you use a blue one? 8. Kiernan. Toluidine blue will stain mast cell granules red. How do basic and acidic dyes bring out the structure of tissues? Name one acidic and one basic dye. J.
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